Preparative Electrophoresis for HDL Particle Size Separation and Intact-Mass Apolipoprotein Proteoform Analysis

Area of Science:

• Lipidomics
• Proteomics
• Cardiovascular Research

Background:

• High-density lipoproteins (HDLs) are crucial for cholesterol transport.
• Apolipoproteins A-I (APOA1) and A-II (APOA2) are key HDL proteins with numerous proteoforms.
• The relationship between HDL proteoform concentrations and particle size remains unclear.

Purpose of the Study:

• To investigate the association between apolipoprotein proteoform concentrations and high-density lipoprotein (HDL) particle size.
• To establish a novel technique for separating HDL particles based on size and analyzing their proteoform composition.

Main Methods:

• Utilized clear native gel-eluted liquid fraction entrapment electrophoresis (CN-GELFrEE) for HDL fractionation.
• Employed mass spectrometry of intact proteins to determine proteoform profiles.
• Used Western blotting to assess molecular diameter of HDL fractions.

Main Results:

• CN-GELFrEE successfully separated HDL particles into 19 and 36 distinct size fractions.
• Fatty-acylated APOA1 proteoforms were significantly associated with larger HDL particles (R = 0.94).
• Acylated APOA1 proteoforms were more abundant in HDL particles >9.6 nm, while HDL-unbound APOA1 lacked acylation.

Conclusions:

• Established CN-GELFrEE as an effective method for separating lipid particles.
• Demonstrated that acylated APOA1 proteoforms are preferentially found on larger HDL particles.
• Suggests a role for APOA1 acylation in determining HDL size and potentially function.